The Dbl-related Protein, Lfc, Localizes to Microtubules and Mediates the Activation of Rac Signaling Pathways in Cells*
- From the ‡Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401, the §Department of Pharmacology and the Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, and the ¶Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia V5Z 4E6, Canada
Abstract
The possibility that the Dbl family member Lfc can activate Rac1 in cells is investigated in this study. Previously, we demonstrated that both Lfc and Lsc, like their closest relative Lbc, can act catalytically in stimulating the guanine nucleotide exchange activity of RhoA in vitro. Neither Lfc nor Lsc stimulated the in vitro exchange activity of Cdc42 or Rac1; however, Lfc was capable of forming a tight complex with Rac1 in vitro. We show here that Lfc stimulates c-Jun kinase (JNK) activity in COS-7 cells. This stimulation was blocked by a dominant negative mutant of Rac1 and somewhat less effectively by dominant negative RhoA, but not by dominant negative Cdc42. Overexpression of Lfc in NIH 3T3 cells induced the formation of actin stress fibers and membrane ruffles, consistent with the activation of both RhoA and Rac1 signaling pathways, whereas overexpression of Lsc led exclusively to well developed stress fibers. Using a recently developed assay for measuring the cellular activation of Rac, we did not find that expression of Lfc increased the levels of GTP-bound Rac1. However, an examination of the cellular localization of Lfc showed that it was localized to microtubules, similar to what has been reported for activated Rac1, the mixed lineage kinase (MLK) and JNK. Moreover, we have found that the Pleckstrin homology (PH) domain of Lfc specifically associates with tubulin. Taken together, these findings suggest a model where the PH domain-mediated localization of Lfc to microtubules enables the recruitment of Rac to a site proximal to its signaling targets, resulting in JNK activation and actin cytoskeletal changes.
Footnotes
-
↵* This work was supported by National Institutes of Health grant GM45478 (to R. A. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵‖ To whom correspondence should be addressed.
- Abbreviations:
- GEF
-
guanine nucleotide exchange factor
- DH
-
Dbl homology
- PH
-
Pleckstrin homology
- Pak
-
p21-activated kinase
- MLK
-
mixed lineage kinase
- JNK
-
c-Jun kinase
- MBP
-
myelin basic protein
- PBD
-
p21 (Cdc42/Rac1) binding domain
- PCR
-
polymerase chain reaction
- PAGE
-
polyacrylamide gel electrophoresis
- GST
-
glutathione S- transferase.
-
- Received July 30, 1998.
- Revision received October 15, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
